利用單分子光鉗直接量測mRAD51組裝至DNA之複合體單元大小

Direct Observation of Mouse RAD51 Assembly Unit Using Optical Tweezers

10.6342/NTU201903151

張晏展

RAD51 ； SWI5-SFR1 ； BRC4 ； 單分子生物物理 ； 光鉗 ； RAD51 ； SWI5-SFR1 ； BRC4 ； single-molecule biophysics ； optical tweezers

國立臺灣大學化學系學位論文

2019年

碩士

李弘文

繁體中文

無論是古細菌、原核或是真核生物的同源重組蛋白，皆在電子顯微鏡下觀察到6-8個單元形成的閉環多聚體，但是實際上在水溶液狀態中更直接的結構資訊則始終不足夠，本實驗利用單分子光鉗以直接的方式量測mRAD51多聚體是否真實存在，且以此單元直接組裝到DNA上。為了精進光鉗系統之穩定度及量測準確度，使的系統解析度足以觀察此單元的即時組裝過程，我們對系統做了以下改進； 1. 將原本以電腦為運算基底的雷射回饋系統改為用電路回饋，有效提升了約一數量級的雷射穩定度。 2. 使用三種獨立的校正方法，準確地決定出光鉗的彈力常數。 3. 以被動式定力鉗取代先前主動式定力鉗的實驗架設，使系統複雜度降低，及提高量測的空間及時間解析度。 4. 利用Allan deviation分析被動式定力鉗穩定度，在46.5 pN情況下，使用一條包含4178 bp雙股和1002 nt單股的DNA，可以在100 ms下得到1 nm的解析度。我們利用被動式定力鉗 (passive force clamp assay)高解析度的直接量測mRAD51與DNA結合過程；在組裝的過程中，可觀察到不連續的、隨機的長度停滯再變長現象，而前後兩個停滯狀態的長度變化，大部分對應到8個mRAD51與DNA的結合。這個結果直接證明了mRAD51與DNA結合的主要單元為8個mRAD51。當加入輔助蛋白mSWI5-SFR1聚合體，結合大小變為以4個mRAD51的結合單元，同時發現核蛋白絲生成速率變為原本之3倍。在加入輔助蛋白mBRCA2之胜肽片段mBRC4後，也降低結合的單元到4個mRAD51並提高核蛋白絲生成速率為原本之5倍。我們的研究顯示，SWI5-SFR1與BRC4可能是以減少RAD51聚合體的大小以及改變RAD51的構型以增加對DNA親和力的方式促進RAD51核蛋白絲的生成。

Recombinases are shown to form closed-ring (hexameric to octameric) structure in electron microscopic studies, but direct structural evidence in solution is missing. We used the passive force-clamp optical tweezers to directly observe the existence of oligomeric recombinase nucleoprotein filament assembly in real-time. To achieve sufficient resolution to resolve individual steps during filament assembly, several instrumental improvements and programming developments have been achieved: (1) improved our home-built optical tweezers stability by using a circuit-based laser feedback to reduce laser power fluctuation; (2) improved the precision and accuracy of trap stiffness calibration by using three independent methods; (3) developed an automatical program to implement passive force clamp; (4) used Allan deviation to validate passive force clamp. Our improved instrument can achieve 1 nm precision in 100 ms using force-activated DNA (a DNA contain 4178 bp dsDNA and 1002 nt ssDNA). We used this improved passive force-clamp optical tweezers to directly observe the assembly dynamics of mouse RAD51 nucleoprotein filament in real-time. Stepwise extension is observed with a single-exponentially distributed dwell between continous extension. Analysis showed that mRAD51 mostly assemble in octamers. Adding accessory proteins, mSWI5-SFR1 complex reduces the assembly unit from octamers to tetramers. Including a short peptide of BRCA2 protein, BRC4, also reduces to tetramers. This reduction in assembly unit is accompanied by increase in extension rates. Our results suggest that the stimulation of mRAD51 filament assembly by mSWI5-SFR1 and mBRC4 are resulted from reduing the assembly unit and increasing the affinity of mRAD51 to DNA.

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